An estimated 170 million people worldwide and 4 million people in the United States are infected with hepatitis C virus (HCV). The majorities of patients do not resolve the infection and develop chronic infections that often lead to end-stage liver disease and hepatocellular carcinoma. Current treatment is limited to a combination therapy of ribavirin and interferon 1. This therapy is expensive and ineffective in 50% of infected individuals. Thus, there is an urgent need to identify viral or cellular molecules that can be used as novel targets in antiviral therapy. It was discovered that HCV binds two molecules of a liver-specific microRNA, miR-122, resulting in a novel, unprecedented upregulation of the viral genome. Sequestration of miR-122 in HCV-infected cultured cells or in livers of infected chimpanzees leads to a dramatic loss of infectious virus without emergence of resistant virus. Therefore, the dependence of HCV on miR-122 presents an Achilles heel of the virus that can be explored for antiviral intervention. This application proposes to study the roles for miR-122 in the viral life cycle and in cholesterol biosynthesis using a novel class of antisense molecules, locked nucleic acids (LNAs) that can easily be delivered to the liver in animals where it sequesters miR-122 in an inactive small duplex RNA. In particular, the first aim will characterize the RNA-RNA interactions in the miR-122/HCV complex, using genetic and biochemical approaches. Aim 2 will test the hypothesis that miR-122 protects the 5' end sequences of the HCV RNA from degradation by ribonucleases or RNA modification enzymes, or aids in the avoidance of activation of double-stranded RNA sensors such as the retinoic acid inducible gene I. These studies will be performed in specific and genome-wide siRNA-mediated gene knockdown experiments. Aim 3 proposes to examine roles for the known isoforms of miR-122 that contain extra 3' terminal adenosine or guanosine residues, on HCV RNA abundance. Deep sequencing analysis and gene knockdown of suspected nucleotidyl transferases will aid in this analysis. The final aim will characterize in detail the mechanism by which miR-122 regulates the expression of Insig1, the major negative regulator of cholesterol and fatty acid metabolism in the liver. In particular, the miR-122-mediated down-regulation of a distinct polyadenylation/cleavage site in a specific Insig1 isoform mRNAs will be examined. Overall, this application will address fundamental aspects about the functions of miR-122 in the HCV life cycle and cholesterol metabolism. The outcomes from these studies will detail novel mechanisms of gene expression mediated by microRNAs in eukaryotic cells and will point to new venues for antiviral therapies.